The proposed research will investigate how protease inhibitors (e.g. PMSF, DFP, TLCK, TPCK) and protease substrates (e.g. TME, TAME) inhibit the binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen and progesterone to intracellular receptors and serum proteins. Protease substrates containing D-amino acids will be used to determine if inhibition of steroid binding to receptors is by action at the site which binds the steroid (competitive inhibition) or if inhibition occurs by an allosteric mechanism (noncompetitive inhibition). Nonradioactive and radioactive PMSF, DFP, TLCK and TPCK will be used to determine the structure of their binding site in rat alpha-fetoprotein and other steroid binding proteins. This structure will be compared with that in serine residue proteases to determine what similarities in mechanism exist between a steroid binding protein and proteases. Steroid binding proteins will be tested for enzymatic activity using sensitive radioactive substrates. Our results with protease substrates will be applied to purifying steroid receptors by affinity columns containing a protease substrate coupled to an agarose matrix. We expect that our studies will be useful in developing techniques for regulating the binding of steroid hormones to their receptors. This should find application in such diverse areas as reproductive biology, control of malignant cell growth and endocrine disorders.